Purification, molecular cloning, heterologous expression and characterization of pig CYP1A2.
Porcine cytochrome P450 (CYP) 1A2 was purified to electrophoretic homogeneity from the hepatic microsomes of beta-naphthoflavone-treated male pigs. In a reconstituted system, this enzyme showed a good catalytic activity towards caffeine, acetanilide, and methoxyresorufin, all known markers of mammalian CYP1A2. Using 3'- and 5'-rapid amplification of coding DNA (cDNA) ends (RACE), we amplified from the liver RNA of control pigs a full-length 1827 bp cDNA containing an open reading frame of 1548 bp which encoded a putative CYP1A2 protein of 516 amino acids and an estimated Mr of 58 380 Da. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that the messenger RNA (mRNA) of CYP1A2 was expressed in liver, heart and nasal mucosa but not in lung, small intestine, kidney and brain. Using the pCW vector containing a N-terminal modified cDNA, pig CYP1A2 was expressed in Escherichia coli. 3-[(3-Chloroamidopropyl)dimethylmmonio]-1-propane-sulfonate (CHAPS)-solubilized E. coli preparations expressing CYP1A2 produced a functionally isoform which, in a reconstituted system, was catalytically active toward ethoxyresorufin and methoxyresorufin showing K(m)'s similar to those obtained with CYP1A2 purified from pig liver or human recombinant CYP1A2. Taken together, these results demonstrate that domestic pigs have a functionally active CYP1A2 gene well expressed in the liver with biochemical properties quite similar to those corresponding to the human enzyme.
Messina
A
A
Istituto di Fisiologia Clinica, Area della Ricerca CNR, Pisa, Italy.
Chirulli
V
V
Gervasi
P G
PG
Longo
V
V
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D034421:Sus scrofa / Q000378:metabolism*
D018076:DNA, Complementary; D019388:Cytochrome P-450 CYP1A2